Microfluidic devices and methods for denudation of cells

Abstract

Disclosed herein are microfluidic devices and methods for denudation of biological materials, such as oocytes and embryos. For example, the devices and methods achieve separation of oocytes from surrounding cumulus cells in a fully automated manner and without manual intervention. The devices can facilitate continuous uni-directional fluid flow, as well as a bi-directional fluid flow mechanism that closely mimics manual methods.

What is claimed is: 
     
         1 . A device comprising:
 (a) a well configured to receive a cell and a fluid at an open upper end, wherein the well is fluidically connected to an outlet channel, wherein the well comprises the open upper end, a lower base, a well outlet, a perimeter, and a denudation cavity;   (b) the outlet channel configured to expel the fluid from the well through the well outlet, wherein the outlet channel comprises an outlet channel inlet and an outlet channel outlet; and   (c) the denudation cavity configured to denude the cell, wherein the denudation cavity protrudes from the perimeter of the well towards the outlet channel.   
     
     
         2 . The device of  claim 1 , wherein the denudation cavity has a dimension that is approximately a diameter of the cell. 
     
     
         3 . The device of  claim 1 , wherein the denudation cavity has two dimensions, wherein each of the two dimensions is approximately a diameter of the cell. 
     
     
         4 . The device of  claim 1 , wherein the denudation cavity is configured to denude the cell by mechanical shear force caused by the cell moving in and out of the denudation cavity. 
     
     
         5 . The device of  claim 4 , wherein the mechanical shear force is lesser than 50 Pa. 
     
     
         6 . The device of  claim 4 , wherein the mechanical shear force is lesser than 10 Pa. 
     
     
         7 . The device of  claim 1 , wherein the device is configured to generate bi-directional flow of the fluid between the well and the outlet channel. 
     
     
         8 . The device of  claim 1 , wherein a cross-sectional area of the outlet channel is dimensioned to lessen a likelihood that the cell passes through the outlet channel. 
     
     
         9 . The device of  claim 1 , wherein the outlet channel is located at the lower base of the well. 
     
     
         10 . The device of  claim 1 , wherein the outlet channel is not located at the lower base of the well. 
     
     
         11 . The device of  claim 1 , wherein the outlet channel is a microfluidic channel. 
     
     
         12 . The device of  claim 1 , further comprising an inlet channel configured to introduce the fluid into the well through a well inlet, wherein the inlet channel comprises an inlet channel inlet and an inlet channel outlet. 
     
     
         13 . The device of  claim 12 , wherein the device is configured to allow bi-directional flow of the fluid between the well, the outlet channel, and the inlet channel. 
     
     
         14 . The device of  claim 12 , wherein a cross-sectional area of the inlet channel is dimensioned to lessen a likelihood that the cell passes through the inlet channel. 
     
     
         15 . The device of  claim 12 , wherein the inlet channel is located at the lower base of the well. 
     
     
         16 . The device of  claim 12 , wherein the inlet channel is not located at the lower base of the well. 
     
     
         17 . The device of  claim 12 , wherein the outlet channel is on a plane that is the same as a plane of the inlet channel. 
     
     
         18 . The device of  claim 12 , wherein the outlet channel is on a plane that is not the same as a plane of the inlet channel. 
     
     
         19 . The device of  claim 12 , wherein the inlet channel is a microfluidic channel. 
     
     
         20 . The device of  claim 12 , further comprising a storage reservoir configured to store the fluid, wherein the inlet channel is configured to introduce the fluid from the reservoir into the well. 
     
     
         21 . The device of  claim 1 , wherein the denudation cavity is configured to denude the cell from cumulus or corona cells. 
     
     
         22 . The device of  claim 1 , wherein the cell is a cumulus oocyte complex. 
     
     
         23 . The device of  claim 1 , wherein the cell is an oocyte comprising cumulus or corona cells on the surface of the oocyte. 
     
     
         24 . The device of  claim 23 , wherein the outlet channel is configured to expel cumulus or corona cells from the well. 
     
     
         25 . The device of  claim 1 , further comprising a waste reservoir configured to store waste, wherein the outlet channel is configured to expel the waste from the well to the waste reservoir. 
     
     
         26 . The device of  claim 25 , wherein the outlet channel is configured to expel cumulus or corona cells from the well into the waste reservoir. 
     
     
         27 . The device of  claim 1 , wherein the fluid is cell media. 
     
     
         28 . The device of  claim 1 , wherein the fluid is embryo culture media. 
     
     
         29 . The device of  claim 1 , wherein the fluid comprises hyaluronidase. 
     
     
         30 . The device of  claim 1 , wherein the well is conical shaped. 
     
     
         31 . The device of  claim 1 , wherein the device is a microfluidic chip. 
     
     
         32 . A method for denuding a cell, the method comprising:
 (a) filling a well of a device with a fluid,   
       wherein the device comprises:
 the well configured to receive the cell and the fluid at an open upper end, wherein the well is fluidically connected to an outlet channel, wherein the well comprises the open upper end, a lower base, a well outlet, and a denudation cavity; 
 the outlet channel configured to expel the fluid from the well through the well outlet, wherein the outlet channel comprises an outlet channel inlet and an outlet channel outlet; and 
 the denudation cavity configured to denude the cell, wherein the denudation cavity protrudes from the perimeter of the well towards the outlet channel; 
 (b) introducing into the upper end of the well the cell; and 
 (c) alternatively applying negative pressure and positive pressure within the device to generate a bi-directional flow of the fluid between the well and the outlet channel to transport the cell in and out of the denudation cavity, thereby denuding the cell. 
 
     
     
         33 . The method of  claim 32 , wherein the alternative application of negative pressure and positive pressure within the device is applied until the cell is completely denuded as determined by visual analysis. 
     
     
         34 . The method of  claim 33 , wherein the visual analysis is by a computer vision algorithm. 
     
     
         35 . The method of  claim 32 , wherein the alternative application of negative pressure and positive pressure within the device is applied for at least 15 minutes. 
     
     
         36 . The method of  claim 32 , wherein the alternative application of negative pressure and positive pressure within the device is applied for at least 30 minutes. 
     
     
         37 . The method of  claim 32 , wherein the alternative application of negative pressure and positive pressure within the device occurs for about 15 minutes to about 30 minutes. 
     
     
         38 . The method of  claim 32 , further comprising priming the well and the outlet channel with the fluid prior to a). 
     
     
         39 . The method of  claim 32 , further comprising retrieving the denuded cell from the upper end of the well. 
     
     
         40 . The method of  claim 32 , wherein the denudation cavity has a dimension that is approximately a diameter of the cell. 
     
     
         41 . The method of  claim 32 , wherein the denudation cavity has two dimensions, wherein each of the two dimensions is approximately a diameter of the cell. 
     
     
         42 . The method of  claim 32 , wherein the denudation cavity is configured to denude the cell by mechanical shear force caused by the cell moving in and out of the denudation cavity. 
     
     
         43 . The method of  claim 42 , wherein the mechanical shear force is lesser than 50 Pa. 
     
     
         44 . The method of  claim 42 , wherein the mechanical shear force is lesser than 10 Pa. 
     
     
         45 . The method of  claim 32 , wherein the device is configured to generate bi-directional flow of the fluid between the well and the outlet channel. 
     
     
         46 . The method of  claim 32 , wherein a cross-sectional area of the outlet channel is dimensioned to lessen a likelihood that the cell passes through the outlet channel. 
     
     
         47 . The method of  claim 32 , wherein the outlet channel is located at the lower base of the well. 
     
     
         48 . The method of  claim 32 , wherein the outlet channel is not located at the lower base of the well. 
     
     
         49 . The method of  claim 32 , wherein the outlet channel is a microfluidic channel. 
     
     
         50 . The method of  claim 32 , wherein the device further comprises an inlet channel configured to introduce the fluid into the well through a well inlet, wherein the inlet channel comprises an inlet channel inlet and an inlet channel outlet. 
     
     
         51 . The method of  claim 50 , wherein the device is configured to allow bi-directional flow of the fluid between the well, the outlet channel, and the inlet channel. 
     
     
         52 . The method of  claim 50 , wherein a cross-sectional area of the inlet channel is dimensioned to lessen a likelihood that the cell passes through the inlet channel. 
     
     
         53 . The method of  claim 50 , wherein the inlet channel is located at the lower base of the well. 
     
     
         54 . The method of  claim 50 , wherein the inlet channel is not located at the lower base of the well. 
     
     
         55 . The method of  claim 50 , wherein the outlet channel is on a plane that is the same as a plane of the inlet channel. 
     
     
         56 . The method of  claim 50 , wherein the outlet channel is on a plane that is not the same as a plane of the inlet channel. 
     
     
         57 . The method of  claim 50 , wherein the inlet channel is a microfluidic channel. 
     
     
         58 . The method of  claim 50 , further comprising priming the well, the outlet channel, and the inlet channel with the fluid prior to (a). 
     
     
         59 . The method of  claim 32 , wherein the device further comprises a storage reservoir configured to store the fluid, wherein the inlet channel is configured to introduce the fluid from the reservoir into the well. 
     
     
         60 . The method of  claim 32 , wherein the denudation cavity is configured to denude the cell from cumulus or corona cells. 
     
     
         61 . The method of  claim 32 , wherein the cell is a cumulus oocyte complex. 
     
     
         62 . The method of  claim 32 , wherein the cell is an oocyte comprising cumulus or corona cells on the surface of the oocyte. 
     
     
         63 . The method of  claim 32 , wherein the outlet channel is configured to expel cumulus or corona cells from the well. 
     
     
         64 . The method of  claim 32 , wherein the device further comprises a waste reservoir configured to store waste, wherein the outlet channel is configured to expel the waste from the well to the waste reservoir. 
     
     
         65 . The method of  claim 32 , wherein the outlet channel is configured to expel cumulus or corona cells from the well into the waste reservoir. 
     
     
         66 . The method of  claim 32 , wherein the fluid is cell media. 
     
     
         67 . The method of  claim 32 , wherein the fluid is embryo culture media. 
     
     
         68 . The method of  claim 32 , wherein the fluid comprises hyaluronidase. 
     
     
         69 . The method of  claim 32 , wherein the well is conical shaped. 
     
     
         70 . The method of  claim 32 , wherein the device is a microfluidic chip.